Polypeptides having polymerase activity and methods of use thereof

Abstract

The invention relates to thermostable polymerases that have polymerase activity temperatures in the range from 90° C. up to 113° C., such as those derived from Pyrolobus fumaria , and to polynucleotides encoding the polymerases In addition, methods of designing new thermostable DNA polymerases and methods of use thereof are also provided. The polymerases have increased activity and stability at increased pH and temperature.

1. A method of preparing cDNA from mRNA, comprising:
 (a) contacting mRNA with an oligo(dT) primer or other complementary primer to form a hybrid; and 
 (b) contacting the hybrid formed in step (a) with a polypeptide encoded by the nucleic acid sequence having the sequence as set forth in SEQ ID NO:3, said polypeptide having polymerase activity, and four different dNTPs, under conditions whereby a cDNA is obtained. 
 
     
     
       2. A method of amplifying a double-stranded DNA molecule comprising:
 (a) providing a first and a second primer, wherein the first primer is complementary to a sequence at or near the 3′-termini of a first strand of the DNA molecule and the second primer is complementary to a sequence at or near the 3′ termini of a second strand of the DNA molecule; 
 (b) hybridizing the first primer to the first strand arid the second primer to the second strand in the presence of a polypeptide encoded by the nucleic acid sequence having the sequence as set forth in SEQ ID NO:3, said polypeptide having polymerase activity, under conditions such that a third DNA molecule complementary to the first strand and a fourth DNA molecule complementary to the second strand are synthesized; 
 (c) denaturing the first and third strands, and second and fourth strands; and 
 (d) repeating steps (a) to (c) one or more times to generate an amplified DNA molecule. 
 
     
     
       3. The method of  claim 2 , further comprising inserting the amplified DNA molecule into a vector. 
     
     
       4. The method of  claim 3 , wherein the vector is a plasmid. 
     
     
       5. The method of  claim 1  wherein the polypeptide has polymerase activity at a temperature in a range from about 90° C. to 113° C. 
     
     
       6. The method of  claim 1  wherein the polymerase activity comprises 3′→45′ exonuclease activity. 
     
     
       7. The method of  claim 1  wherein the polypeptide has polymerase activity under conditions of high or low salinity. 
     
     
       8. The method of  claim 1  wherein the polypeptide has polymerase activity in the presence of organic solvents. 
     
     
       9. The method of  claim 2  wherein the polypeptide has polymerase activity at a temperature in a range from about 90° C. to 113° C. 
     
     
       10. The method of  claim 2  wherein the polymerase activity comprises 3′→5′ exonuclease activity. 
     
     
       11. The method of  claim 2  wherein the polypeptide has polymerase activity under conditions of high or low salinity. 
     
     
       12. The method of  claim 2  wherein the polypeptide has polymerase activity in the presence of organic solvents. 
     
     
       13. A method of generating a polynucleotide comprising:
 (a) providing a nucleic acid primer complementary to a sequence in the polynucleotide; 
 (b) contacting and hybridizing the primer to the polynucleotide in the presence of a polymerase under conditions such that a second nucleic acid having a sequence complementary to the polynucleotide is generated, wherein the polymerase comprises the amino acid as set forth in SEQ ID NO:4. 
 
     
     
       14. The method of  claim 13 , wherein the nucleic acid primer further comprises a label. 
     
     
       15. The method of  claim 14 , wherein the label comprises a radioactive isotope. 
     
     
       16. The method of  claim 14 , wherein the label comprises a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a metal chelator or an enzyme. 
     
     
       17. The method of  claim 13 , wherein conditions in step (b) comprise contacting the primer to the polynucleotide in the presence of the polymerase in the presence of non-natural nucleotides or nucleotide analogs. 
     
     
       18. The method of  claim 17 , wherein the non-natural nucleotides or nucleotide analogs comprise an inosine, a 2-aminopurine, a 5-methylcytosine, an [α]dATP, a 7-deaza-dGTP or a 7-deaza-dATP. 
     
     
       19. The method of  claim 17 , wherein the non-natural nucleotides or nucleotide analogs comprise a dideoxyribonucleoside triphosphate (ddNTP). 
     
     
       20. The method of  claim 17 , wherein the dideoxyribonucleoside triphosphate (ddNTP) comprises ddATP, ddCTP, ddGTP, ddTTP or ddTTP. 
     
     
       21. The method of  claim 13 , wherein the method comprises a PCR amplification reaction. 
     
     
       22. A method of amplifying a double-stranded DNA molecule comprising:
 (a) providing a first and a second primer, wherein the first primer is complementary to a sequence at or near the 3′-termini of a first strand of the DNA molecule and the second primer is complementary to a sequence at or near the 3′ termini of a second strand of the DNA molecule; 
 (b) hybridizing the first primer to the first strand and the second primer to the second strand in the presence of a polypeptide having polymerase activity encoded by the nucleic acid comprising the sequence as set forth in SEQ ID NO:3, or an enzymatically active fragment thereof, wherein the polypeptide has polymerase activity at a temperature in a range from about 100° C. to 107° C., under conditions such that a third DNA molecule complementary to the first strand and a fourth DNA molecule complementary to the second strand arc synthesized; 
 (c) denaturing the first and third strands, and second and fourth strands; and 
 (d) repeating steps (a) to (c) one or more times to generate an amplified DNA molecule. 
 
     
     
       23. A method of preparing cDNA from mRNA, comprising:
 (a) contacting mRNA with an oligo(dT) primer or other complementary primer to form a hybrid; and 
 (b) contacting the hybrid formed in step (a) with (i) a polypeptide having polymerase activity encoded by the nucleic acid comprising the sequence as set forth in SEQ ID NO:3, or an enzymatically active fragment thereof, and (ii) four different dNTPs, under conditions whereby a cDNA is obtained. 
 
     
     
       24. A method of amplifying a double-stranded DNA molecule comprising:
 (a) providing a first and a second primer, wherein the first primer is complementary to a sequence at or near the 3′-termini of a first strand of the DNA molecule and the second primer is complementary to a sequence at or near the 3′ termini of a second strand of the DNA molecule; 
 (b) hybridizing the first primer to the first strand and the second primer to the second strand in the presence of a polypeptide having polymerase activity encoded by the nucleic acid having the sequence as set forth in SEQ ID NO:3, or an enzymatically active fragment thereof, under conditions such that a third DNA molecule complementary to the first strand and a fourth DNA molecule complementary to the second strand are synthesized; 
 (c) denaturing the first and third strands, and second and fourth strands; and 
 (d) repeating steps (a) to (c) one or more times to generate an amplified DNA molecule.